Antimicrobial composition of a polymer and a peptide forming amphiphilic helices of the magainin-type

ABSTRACT

PCT No. PCT/US94/07019 Sec. 371 Date Dec. 18, 1995 Sec. 102(e) Date Dec. 18, 1995 PCT Filed Jun. 22, 1994 PCT Pub. No. WO95/00547 PCT Pub. Date Jan. 5, 1995Novel polymer-bound oligopeptides exhibiting antimicrobial activity have been develop. The oligopeptides are unique amino acid sequences that form amphiphilic helices.

CROSS-REFERENCE

This case is the National Stage of International Application No.PCT/US94/07019, filed Jun. 22, 1994, under 35 USC 371, aContinuation-In-Part of U.S. application Ser. No. 08/082,852, filed Jun.22, 1993, now abandoned, benefit of which is claimed.

FIELD OF INVENTION

The invention relates to the design and synthesis of novel antimicrobialcompositions comprising unique amino acid sequences that formamphiphilic helices and possess antimicrobial properties. Morespecifically, the invention provides the peptides of amino acidsequences KGLKKLLKLLKKLLKL,(SEQ ID NO:1) LKLLKKLLKL LKKLGK(SEQ ID NO:2)KGGLKKLLKLLKKLLKL (SEQ ID NO:3), LKLLKKLLKLLKKLGGK (SEQ ID NO:4) andKGGGLKKLLKLLKKLLKL (SEQ ID NO:5) LKLLKKLLKLLKKLGGGK (SEQ ID NO:6) wherethese peptides have the capability of killing microorganisms withoutbeing indiscriminately cytotoxic. The invention further relates to theincorporation of these and other antimicrobial peptides into polymercompositions without limiting the bioactivity of the peptides.

BACKGROUND OF THE INVENTION

Many vertebrates and invertebrates secrete natural substances thatpossess both antibacterial and/or indiscriminate cytotoxic properties.Examples of some of these substances include PGLa (frog skin), defensins(human phagocytes), cecropins (Silkmoth pupae or pig intestine),apidaecins (honeybee lymph), melittin (bee venom), bombinin (toad skin)and the magainins (frog skin). Purification of the active constituentsof these natural substances have shown that they consist primarily ofprotein and it has been suggested that they may constitute a system ofcellular immunity in the producing organism.

Peptides and oligopeptides that have activity against microorganismsspan a broad range of molecular weights, secondary conformations andsites of action. Biological activity can range from being specificallybactericidal or fungicidal to being indiscriminately cytotoxic (celllytic) to all cells. Peptides that are specifically bactericidal includelarge polypeptides such as lysozyme (MW 15000 daltons) and attacins (MW20-23,000 daltons) as well as smaller polypeptides such as cecropin (MW4000 daltons) and the magainins (MW 2500 daltons). The spectrum ofbiocidal activity of these peptides is somewhat correlated to size. Ingeneral, the large polypeptides are active against limited types andspecies of microorganisms (e.g., lysozyme against only gram positivebacteria), whereas many of the smaller oligopeptides demonstrate a broadspectrum of antimicrobial activity, killing many species of both grampositive and gram negative bacteria.

Although few similarities exist between the amino acid sequences of thebiocidal peptides, it has been shown that magainin, cecropins, andbombinin oligopeptides form similar secondary structures described as anamphiphilic helix (Kaiser et al. Annu. Rev. Biophys. Biophys. Chem 16,561-581, 1987). Amphiphilic or amphipathic helices are secondary proteinstructures possessing an overall affinity for hydrophillic materials. Ithas been suggested that the affinity of these helices for cell membranesmay account, in part, for their biological activity and the literaturewould suggest that there may be a strong correlation between thissecondary peptide structure and biological activity (Lee et al. Biochem.Biophys. Acta 862, 211-219, (1986)).

One of the first biocidal oligopeptides to be isolated from naturalsources was bombinin and is described by Csordas et al. (Proc. Int.Symp. Anim. Plant Toxins, 2, 515-523, (1970)). Bombinin is found in toadskin secretions and has both antimicrobial and cytotoxic properties.Csordas teaches significant sequence homology between bombinin andmelittin, another antimicrobial peptide, isolated from bee venom.

DeGrado and Kezdy (J.A.C.S. 103, 679-681,(1981)) describe thepreparation of potent synthetic analogues of melittin using rationalpeptide design. The peptide sequences of DeGrado were shown to have bothstructural and functional similarities to melittin in that both attainedsimilar amphiphillic helical secondary conformation and both wereindiscriminately cytotoxic. Subsequently DeGrado et al. (Peptides:Structure and Function., Proc. 8th Amer. Peptide Symp., V. J. Hruby Ed.,(1983)) prepared a series of idealized synthetic oligopeptides to mimicthe cecropins. These cecropins analogues were Fmoc blocked peptidescontaining Leu and Lys repeats and mimicked the bioactivity of theirnatural counterparts.

Zasloff et al. (WO 9004408) describe the use of the amphiphilic formingpeptide, magainin and magainin fluoride analogues as pharmaceuticalcompositions. The peptides of Zasloff are limited in size to between 16and 50 amino acids, and consist of blocks of four amino acids, eachblock containing at least one hydrophobic, one hydrophillic neutral, andone hydrophillic basic amino acid.

Cuervo et al. (WO 9006129) describe the preparation of deletionanalogues of magainin I and II for use as pharmaceutical compositions.They disclose a general scheme for the synthetic preparation ofcompounds with magainin-like activity and structure.

From the art it would appear that biological activity of these peptidesis not only dependent on secondary structure but also is related tosequence length. For most peptides there exists an amino acid minimumbelow which antimicrobial activity is reduced or eliminated. Thesequences of Cuervo (WO 9006129), for example, contain a minimum of nineunique amino acids for antimicrobial activity. Bomen et al. in U.S. Pat.No. 5,096,886 describe a natural cecropin isolated from pig intestinethat contains a minimum of twelve unique amino acids. A minimum ofthirteen unique amino acids are present in the sequence for theantimicrobial polypeptide secreted from Sarcophaga peregrina embryo asdescribed by Natori in U.S. Pat. No. 5,008,371.

Although the antimicrobial and cytotoxic activities of the abovementioned preparations are good, they all consist of proteins that mustbe isolated and purified from natural sources. The isolation of largequantities of these peptides necessary for pharmaceutical and relateduses is both impractical and expensive. Furthermore, many of thesynthetic analogues described above contain a diversity of amino acidsand the synthesis of such molecules is not trivial. Even though blockingstrategies are improving in the art of peptide synthesis, theincorporation of certain amino acids (i.e., Histidine) still poses somedifficulty in solid phase peptide synthesis (Stewart et al. Solid PhasePeptide Synthesis, 2nd Edit, J. M. Stewart, J. D. Young, 1984, PierceChem. Co. pp 18-27). Histidine is one of the unique amino acids in thecore sequence described by Cuervo and Houghten (WO 9006129).

Lee et al. Biochem. Biophys. Acta 862, 211-219, (1986) describe thepreparation of basic model synthetic peptides containing highlyconserved amino acid sequences possessing antimicrobial activityspecifically against gram positive bacteria. The peptides of Lee containmixtures of Leu, Ala, Lys and Arg. The antimicrobial activities of thesepeptides roughly parallel their alpha helix content. Bioactivity ofthese peptides may be further correlated with the position of the basicand hydrophobic groups in the sequence. Thus high alpha-helix contentcorresponds to high biological activity and peptides where thehydrophobic groups and the hydrophilic cationic groups are segregated onthe face of the helix, exhibit the most potent biological activity. Leefurther teaches that the primary requirement for biological activity isthe structure-forming potential of the sequence in a hydrophobicenvironment.

Although the peptides of Lee appear to be selectively active againstgram positive bacteria as opposed to gram negative bacteria, Lee alsoteaches that synthetic sequences with Arg or Lys between short patchesof hydrophilic amino acids cause leaking from mitochondrion, microsomes,lysosomes and red blood cells suggesting that these peptides do nothighly discriminate among structures containing lipid bilayer membranesurfaces.

Suenaga et al. (Biochem. Biophys. Acta 981, 143-150, (1989)) teach thatblocks of four, 4-mer peptides of the same or similar composition tothose of Lee are capable of forming amphipathic helices and are usefulin inducing the fusion of unilamellar lipid vesicles. These peptideswere seen to cause extensive perturbation of the lipid bilayer prior tofusion, again emphasizing the interaction of these peptides with lipidbilayer structures.

Houghten et al. (WO 9201462) advanced the earlier work of Lee andSuenaga teaching oligopeptides which have activity against both grampositive and gram negative bacteria without being indiscriminatelycytotoxic. The model oligopeptide of Houghten consists of a sequencethat contains only Lys and Leu amino acids of the formula SEQ ID NO:7.Houghten teaches that there is no antimicrobial activity when the lengthof the peptide exceeds 25 amino acids or contains less than 8 aminoacids and that she most active sequences contain hydrophilic content ofbetween 50 and 60%. Furthermore, when the charge content of the sequenceexceeds 54% the oligopeptide loses potent activity against gram negativebacteria even though an amphiphillic secondary structure is maintained.

Buttner et al. (Biopolymers 32, 547-583, (1992)) describe therelationship between antimicrobial activity and alpha helix content of apeptide. Buttner teaches that preservation of the hydrophobic side ofthe helix is important to the potency of the antimicrobial activity.Thus, it would appear that antimicrobial peptides as taught by Lee orHoughten must additionally have a proper balance of hydrophobic andhydrophilic amino acids and these amino acids must be judiciously placedin the sequence such that an alpha helix secondary structure can beachieved in the presence of a hydrophobic environment.

The peptides described above are useful, however, each have significantdrawbacks. The peptides of Lee, although synthetic and containing asimpler composition than those isolated from nature, still contain asignificant percentage of Ala and Arg which contribute to the complexityof the synthesis of the sequence. Although the simpler peptides ofHoughten contain only Leu and Lys they require a relatively highpercentage of the basically charged amino acid Lys. The requirement fora high percentage of Lys limits these sequences in light of the furthernecessity of having peptides with a basic charge content of less than54% to maintain biological activity.

WO-A-92/2317 discloses amino acid compositions comprising a C-terminalsubstituted peptide of the formula X-CO-T where X is an amphiphilicpeptide and T comprises a variety of substituents.

There remains a need, therefore, for an antimicrobial peptide of simplecomposition, active against both gram negative and gram positivebacteria that contains a low percentage of basic charged amino acidsthat lends itself to production in commercial quantities.

The composition of the present invention represents an improvement overthe prior art, providing a sequence that is simple and adaptable forcommercialization, that demonstrates activity against both gram positiveand negative bacteria without being indiscriminately cytotoxic, and thathas a lower Lys (or basic, charged amino acid) content than thesequences taught in the art. The lower Lys content of the sequences ofthe instant invention render them more versatile, permittingsubstitution of other hydrophilic amino acids for Lys without decreasingbioactivity.

The expanding needs in the medical and fabrics industries formaintaining sterile and odor free environments has led to thedevelopment of materials with inherent antimicrobial properties. For themost part, polymers and polymer compositions have been targeted forenhancement with antimicrobial agents. Methods of rendering polymersantimicrobial are varied, and include the incorporation of metals andmetal ions into the polymer, the use of various polymer coatings such asalpha or beta chitins and metal containing zeolites, or irradiation ofpolymer fibers to produce surface active antimicrobial groups such asquaternary or tertiary amines (White et al., Chemically ModifiedSurfaces, Vol. 1 Silanes, Surfaces & Interfaces, D. E. Leyden, Edit.1985, 107-140. and Goldstein, L, Journal of Chromatoaraphy, 215, 31,(1981)). The above methods all rely on reactive groups on the surface ofthe polymer to provide the antimicrobial function.

Alternatively, antimicrobial polymers have been designed where theantimicrobial moiety is released from the polymer surface. Shiraishi etal. (J. Macromol Sci-Chem A25 (8) 1015-1025, (1988)) describe thepreparation of polymer derivatives of chloramphenicol which acts as aninhibitor of protein synthesis. Chloramphenicol release from thepolymeric precursor prodrug is required for activity againstmicroorganisms. Dumitriu et al. (Coll & Poly. Sci. 267:595-599 (1989))describe bioactive polymers with coupled chloramphenical and ampicillin.Singh et al. (Die Angew. Makromol. Chemie, 172 87-102, (1989)) describea method for radiation grafting of methacrylic acid onto silk toimmobilized 8-hydroxyquinoline HCl. The drug is immobilized solely byelectrostatic interaction and the antimicrobial activity is a result ofsustained release of the drug into the media.

Formulas with unusual peptides and amino acids have been used incontrolling the infection or growth of fungi on a variety of articles.For example, Schmatz et al. (GB 2241955) describe a cyclic hexapeptidecontaining hydroxy groups. Sato et al. (GB 2099301) describe the coatingof synthetic fibers with solutions containing an amino acid surfactantcontaining a C8 to C16 fatty acid. The surfactant is comprised ofnon-natural amino acids and has a relatively low antimicrobial specificactivity. Additionally the fatty acid constituents have a negativeimpact on the water solubility of the polymer compound. Bunyan (BE844904) describes the preparation of surgical dressing consisting of afilm-forming protein and its treatment with hypochlorite to create abactericidal. It is the Hypochlorite and not the polypeptide that is thedisinfecting agent in the compositions of Bunyan. Unitika (JP 02091275)teaches the coating of a formed yarn with an antibacterial polyaminoacid. Besides being antimicrobial the incorporation of the polyaminoacid imparts a coating with a "softer touch" quality to the polymer.

The above mentioned amino acid containing antimicrobial polymers areuseful, but are limited in that the bioactive component must be releasedfrom the fiber to effect antimicrobial activity. The leaching ofbioactive moieties poses the potential problem of contaminating othermaterials with unwanted antimicrobial agents as well as limiting theeffective life of the treated polymer. A preferred polymer compositionwould comprise an antimicrobial peptide chemical bonded to the polymer.Such a composition would represent an improvement over the art as itwould overcome the problem of leaching of bioactive agents while at thesame time possesing extended bioactive utility.

Kaplan et al. (U.S. Pat. No. 5,019,093) discloses a braided suturecomprised of bioabsorbable polymeric materials which exhibits enhancedflexibility and hand. The suture may be mixed with a variety ofantimicrobial aqents including magainin to convey antimicrobialproperties to the suture.

Calcaterra et al. (U.S. Pat. No. 4,810,567) describe the preparation ofantimicrobial fabrics by irradiation of a vinyl-modified polymyxin ontofabrics of a variety of chemical composition. The resultantantimicrobial polymer is active due to the covalent bonding of polymyxinto the fabric. A hydrolyzable material would be detrimental to thisinvention. This particular cyclic peptide antibiotic is not selectiveand according to Franklin and Snow (Biochemistry of AntimicrobialAction, 4th Edition, Chapman & Hall, London 1989, pp. 61-62) thepolymyxins have minor medicinal value because they also target mammalianmembranes. In addition, the process of Calcaterra et al. is fairlynon-selective and does not ensure that the cyclic peptide will beattached to the surface in a manner that optimally presents theantibiotic to the outer membrane. Finally, polymyxins are not veryactive against gram positive bacteria. The classes of antimicrobialsdescribed in U.S. Pat. No. 4,810,567 do not include the antimicrobialpeptides described herein.

Recent advances in peptide synthesis and recombinant genetics have madeit both feasible and practical to use natural and synthetic peptides asbioactive agents in polymer compositions. Polymers with covalentlybonded peptides or polypeptides are known. For example, RGD-containingsequences have been covalently incorporated into polyurethanes (Lin etal., J. Biomater. Sci. Polym. Ed. 3, 217-227, (1992)) for the purpose ofproviding biomaterials for therapeutic use. The RDG peptide sequencesmay be incorporated either as side chains or directly into the polymerbackbone and still retain bioactivity. The principle function of the RDGand other peptide sequences to facilitate is the selective adhesion andgrowth of mammalian cells such as endothelial cells on the surface ofthe polymer. Additionally there are numerous examples of the covalentbinding of larger polypeptides, proteins and enzymes to polymers whichfunction as immobilized biocatalysts .

Recently Duran, L. W., et al., (Soc. Biomater., 19th Annual Meeting,April-May Vol. 26, p. 35 (1993)) have disclosed the covalentphotochemical immobilization of magainin on silicon rubber. It wasdemonstrated that the photoderivatized magainin was active against bothgram positive and gram negative bacteria and that the siliconrubber-peptide composition was active against S. epidermidis. Theteaching of Duran et al., show that, in one instance, bioactive magaininmay be covalently linked to a rubber polymer, however, do not teach themethod of that attachment nor the covalently linking of any otherantimicrobial peptides to polymers. Furthermore, Duran et al., teachcompositions with very low bioactivities making it unlikely that suchcompositions would be practical in commercial applications.

The present invention provides a polymer composition comprising abioactive covalently bonded antimicrobial peptide. The covalent bondedantimicrobial peptide polymer is regenerable after use and thus hasconsiderably extended lifetime over the such systems whose mechanism ofaction relies on the leaching or hydrolysis of the bioactive peptideinto the soluble fluids.

SUMMARY OF THE INVENTION

The present invention provides a series of non-natural oligopeptidesuseful as antimicrobial agents. These oligopeptides are related in thatthey share a common amino acid sequence, referred to as the coreoligopeptide. The core oligopeptide has antimicrobial activity againstgram negative and gram positive bacteria and against yeasts. Moreparticularly, this invention provides analogues of the coreoligopeptide, wherein at least one amino acid residue has been added toeither the amino terminus or the carboxy terminus of the coreoligopeptide chain, where additions to the amino terminus are prefered.The preferred analogues are referred to as N-addition analogues andtheir sequences are given, along with the core oligopeptide in Table I.The N-addition analogues have greater antimicrobial activity than thecore oligopeptide. The oligopeptides of the present invention have anamphiphilic helical secondary structure resembling naturally occurringantimicrobial peptides and the shape is considered essential toantimicrobial activity.

                                      TABLE I                                     __________________________________________________________________________    Sequences of Non-Natural Oligopeptides                                        with Antimicrobial Activity                                                   Formula                                                                           Amino Acid #                                                              #   -4 -3 -2 -1 1  2  3  4  5  6  7  8  9  10 11 12 13 14                     __________________________________________________________________________    I               Leu                                                                              Lys                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu                                                                              (SEQ ID                                                                        NO:8)              II        Lys                                                                              Gly                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu (SEQ ID                                                                       NO:1)              III    Lys                                                                              Gly                                                                              Gly                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu (SEQ ID                                                                       NO:3)              IV  Lys                                                                              Gly                                                                              Gly                                                                              Gly                                                                              Leu                                                                              Lys                                                                              Lsy                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Leu                                                                              Leu                                                                              Lys                                                                              Leu (SEQ ID                                                                       NO:5)              __________________________________________________________________________

The present invention further provides a biologically active amphiphilicoligopeptide, said oligopeptide being of the general formula V: FormulaV:

(R--Cx)y-(ABBAABA)z, wherein:

x=0, 1, 2, or 3;

y=0,1;

z=>1;

R=X--(CH2)n--CH(Y)--C(O) where n=1-6;

X=--NH2, NH2--C(O)--NH--C(NH2)═NH, or --C(NH2)═NH;

Y=X, H or --NH(Bl) where Bl is a common blocking group such as acetyl;

A=Leu, Met, Phe, Ala, Val or Ile;

B=Lys, Arg, His, Gln, Asn, Ser, Thr and Orn (Ornithine); and

C=Gly or Ala.

The instant invention further provides a polymer-oligopeptidecomposition comprising of a polymer, and an oligopeptide of formula Vwherein the polymer is selected from the group consisting ofpolyurethane, polyetherurethane, polyester, silicone, polyamide,polyolefin, polypeptide, polysaccharide, cellulosic, or silk. Thepolymer-oligopeptide compositions may comprise the oligopeptide coatedon the surface of the polymer or covalently incorporated in the polymeras side chain moieties. The oligopeptide is not consumed by themicrobial cell and therefore may act many times. Consequently lesspeptide is necessary as part of the composition and there is less chancethat mircoorganisms will develop resistance to these oligopeptides.

Also provided is a process for inhibiting the growth of microorganismcomprising the steps of: contacting an effective amount of apolymer-oligopeptide composition with a population of microorganisms;and leaving the polymer-oligopeptide in contact with the microorganismpopulation for a time sufficient to inhibit microorganism growth.

Additionally a process is provided for the production of anantimicrobial oligopeptide-polymer composition comprising the steps of:modifying a suitable polymer by the addition of a non-clevable linkergroup; synthesizing an Fmoc blocked peptide using Fmoc blocked aminoacids and the techniques of solid phase peptide synthesis wherein thefirst amino acid of the peptide to be synthesized is joined to thenon-clevable linker; and deprotecting the Fmoc blocked peptide.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEOUENCE LISTINGS

FIG. 1 illustrates a general scheme for stepwise solid phase peptidesynthesis, where R=insoluble polymeric support; AA1, AA2 . . . AAn=aminoacid residues numbered starting from C-terminus; T="temporary"protection; ◯="permanent" protection over the course of peptidesynthesis; =free carboxyl; =free amino

FIG. 2 illustrates reactions for synthesis of monoamine and bisamineoligopeptides derivatives.

FIG. 3a and 3b illustrate a general reaction method for the covalentattachment of oligopeptides corresponding to formulas I-IV into apolyamine polymer.

FIG. 4a illustrates cell growth inhibition of E. coli by the coreoligopeptide corresponding to formula I.

FIG. 4b illustrates cell growth inhibition of E. coli by the N-additionanalogue corresponding to formula II.

FIG. 4c illustrates cell growth inhibition of E. coli by the N-additionanalogue corresponding to formula III.

FIG. 4d illustrates cell growth inhibition of E. coli by the N-additionanalogue corresponding to formula IV.

FIG. 5 illustrates cell growth inhibition of E. coli by a silk-coreoligopeptide composition.

Applicant provides Sequence Listings 1-18 in conformity with "Rules forthe Standard Representation of Nucleotide and Amino Acid Sequences inPatent Applications" (Annexes I and II to the Decision of the Presidentof the EPO, published Supplement No. 2 to OJ EPO 12/1992).

DETAILED DESCRIPTION OF THE INVENTION

As used herein the following terms may be used for interpretation of theclaims and specification.

The terms "peptide" and "oligopeptides" will be used interchangably andwill refer to amino acid sequences of between two and thirty amino acidsin length.

The term "core oligopeptide" will refer to peptides given by the formula(Leu-Lys-Lys-Leu-Leu-Lys-Leu)_(n) (SEQ ID NO:9) wherein the amino acidsequence occurs from left to right as shown or from right to left andwhere n=1, 2, or 3. The core oligopeptide will also be indicated by theabbreviation "LKP".

The term "antimicrobial" means as having to do with the killing orgrowth inhibition of microbial organisms.

The term "cytotoxic" means the killing or lysis of eukaryotic organisms.

Ther terms "amphiphillic helix" and "amphipathic helix" are usedinterchangably and mean any protein or peptide secondary structure thatforms a helix wherein that helix includes both hydrophobic andhydrophilic regions and demonstrates an affinity for hydrophillicstructures such as those found in lipid bilayers and cell membranes.

The term "class A amino acids" refers to those amino acids with a nethydrophobic affinity.

The term "class B amino acids" refers to those amino acids with a netnegative electrostatic charge.

The term "class C amino acids" refers to amino acids selected from thegroup consisting of Gly or Ala.

The term "N-addition analogue" refers to any derivative of the coreoligopeptide amino acid sequence where at least one amino acid is addedto either the N-terminal or the carboxy end of the core oligopeptidewhere additions to the amino terminus are preferred. The amino acidadditions to the core oligopeptide may be of any type being eithernatural or non-natural, hydrophobic, hydrophilic or basic. The preferredN-addition analogues of the instant invention will be abbreviated SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5 corresponding to the formulae II,III, and IV of Table I respectively.

The terms "side chain blocking group" or "side chain protecting group"refers to labile chemical groups that are covalently attached to achemically-reactive site on the amino acid. The blocking or protectinggroup acts to prevent chemically-reactive sites from competing orinterfering with the desired specific coupling of an alpha amino groupof one amino acid to a carboxyl group on another amino acid to form apeptide bond. The blocking or protecting group is labile to allowselective removal under appropriate conditions. Common blocking groupsmay include, but are not limited to acetyl (Ac), trifluoroacetyl (Tfa),tert-butyloxy-carbonyl (Boc), benzoyl (Bz), and9-fluorenylmethyl-oxycarbonyl (Fmoc).

The term "non-cleavable linker" refers to any chemical moiety orcompound capable of modifying a suitable polymer through which acovalent attachment of an oligopeptide may be accomplished. A typicalexample of a non-cleavable linker is ethylenediamine (EDA).

The term "MIC" refers to minimal inhibitory concentration and will bedefined as the lowest concentration of either soluble peptide or peptideimmobilized on a polymer that results in total kill of bacteria.

The term "DIC" refers to the compound diisopropyl carbodiimide.

The term "DMAP" refers to the compound dimethylaminopyridine.

The term "NMM" refers to the compound N-methyl-morpholine.

The term "DCM" refers to the compound dichloromethane.

The term "DMF" refers to the compound dimethylformamide.

The term "DIEA" refers to the compound N,N'-diisopropylethylamine.

The term "Castro's reagent" and "BOP" are used interchangeably and aredefined as the mixture of compounds comprising(benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate).

As used herein the following abbreviations will be used to identifyspecific amino acids:

    ______________________________________                                                           Three-Letter                                                                             One-Letter                                      Amino Acid         Abbreviation                                                                             Symbol                                          ______________________________________                                        Alanine            Ala        A                                               Arginine           Arg        R                                               Asparagine         Asn        N                                               Aspartic acid      Asp        D                                               Asparagine or aspartic acid                                                                      Asx        B                                               Cysteine           Cys        C                                               Glutamine          Gln        Q                                               Glutamine acid     Glu        E                                               Glutamine or glutamic acid                                                                       Glx        Z                                               Glycine            Gly        G                                               Histidine          His        H                                               Leucine            Leu        L                                               Lysine             Lys        K                                               Methionine         Met        M                                               Phenylalanine      Phe        F                                               Proline            Pro        P                                               Serine             Ser        S                                               Threonine          Thr        T                                               Tryptophan         Trp        W                                               Tyrosine           Tyr        Y                                               Valine             Val        V                                               ______________________________________                                    

Peptides of the present invention are effective as antibiotics and canbe employed to inhibit, prevent or destroy the growth or proliferationsof microorganisms such as Gram-positive and Gram-negative bacteria,fungi and yeasts.

The peptides can also be used as preservative or sterilants for materialsusceptible to microbial contamination.

The peptides of the present invention can be administered to a targetcell or host by direct or indirect application. For example, the peptidemay be administered topically or systemically.

The peptides of the present invention may be incorporated into films orspinnable fibers for their use in sterilants of material susceptible tomicrobial contamination.

The covalently bonded peptide-polymer compostions of the instantinvention may be used as therapeutics or prophylactics on surfaces wheremicrobial contamination is a problem such as delivery vehicles forfoods, seeds, wound dressings, sutures, catheters, dialysis membranes,water filters, carpets and surgical gowns. Additionally the propertiesof this compostion are useful for both short term and long-term dwellingarticles such as in fabrics, carpets, and catheters. Peptide design:

The present invention provides a series of non-natural oligopeptidesuseful as antimicrobial agents having in common the core oligopeptide.The core oligopeptide may be given by the formula:

    (Leu-Lys-Lys-Leu-Leu-Lys-Leu).sub.n                        (SEQ ID NO: 9)

wherein the amino acid sequence occurs from left to right as shown orfrom right to left and where n =1, 2, or 3. Core oligopeptides haveantimicrobial activity against gram negative and gram positive bacteriaand against yeasts but are not indiscriminately cytotoxic. It ispreferred that the core oligopeptide be composed of amino acids witheither an all D- or all L-configuration. The directionality of thehandedness of the resulting helical D and L peptides are opposing,however, the antimicrobial activity of the peptides are identical toeach other. It is also contemplated that introduction of a small numberof D-amino acids into the right-handed alpha helical peptides or a smallnumber of L-amino acids into the predominantly D-amino acid, left-handedhelices would be possible without significantly altering the biologicalactivity of the composition.

The length of the core oligopeptide is variable in this invention. Thecore oligopeptide may be as short as seven amino acids and as long astwenty-one, where a length of at least fourteen amino acids ispreferred.

The core oligopeptide comprises both hydrophobic (hereinafter referredto as class A amino acids) and basic amino acids (hereinafter referredto as class B amino acids) where an excess of hydrophobic amino acids ispreferred and where a ratio of hydrophobic to basic amino acids of about4:3 is most preferred. In a preferred embodiment the core oligopeptidecontains at least fourteen amino acids where eight of the amino acidsare hydrophobic and six amino acids are basic amino acids.

It will be appreciated by one of skill in the art that variants of thecore oligopeptide are possible where suitable hydrophobic, hydrophillicand basic amino acids may be substituted for those of the presentsequence with negligible affect on the bioactivity of the peptide. Forthe purpose of the such substitutions suitable class A amino acids maybe selected from the group comprising Leu, Met, Phe, Ala, Val and Ilewhere Leu is most preferred. Suitable class B amino acids may beselected from the group comprising Lys, Arg, His, Gln, Asn, Ser, Thr andOrn (Ornithine), where Lys is most preferred.

The oligopeptide chain of the core oligopeptide is composed of blocks ofthree and occasionally two amino acids. Each block consists of at leastone class A and one class B amino acid where the third amino acid in thegroup may be of class A or class B and where it is preferred that thethird amino acid lie adjacent to the amino acid of its class (e.g., AABor BAA or ABB or BBA). In designing variants of the core oligopeptidethe groups of three amino acids should be placed next to each other inthe oligopeptide such that the class A amino acids are in pairs and eachpair of class A amino acids is spaced by at least one class B aminoacid, but preferably two B amino acids (e.g., ABBAABAABBAABA;AABBAABABBAABA).

The invention further provides sequences derived from the coreoligopeptide (hereinafter referred to as N-addition analogues) where thecore oligopeptide is extended at either the amino terminus or thecarboxy terminus with at least two and up to four amino acids. For thepurposes of the present invention N-addition analogues comprising theaddition of amino acids at the N-terminus of the core oligopeptide arepreferred.

The N-terminal amino acid of the N-addition analogue may be any naturalor non-natural amino acid that has the formula R=X--(CH2)n--CH(Y)--C(O)where n=1-6; X=--NH2, NH2--C(O)--NH--C(NH2) ═NH, or --C(NH2)═NH; andY=X, H or --NH(Bl) where Bl is a common blocking group. However, for thepurposes of the present invention the N-terminal amino acid mostpreferred is Lys. The amino acids which intervene between the N-terminalLys and the core oligopeptide will be referred to as class C amino acidsand are selected from the group comprising Gly or Ala. The generalsequence formula corresponding to these antimicrobial N-additionanalogues is (R--Cx)y-(ABBAABA)z where x =0, 1, 2, or 3 and y=0,1 andz=>1. The N-addition analogues described by formulae II, III, and IV(Table I) are most preferred. As with the core oligopeptide thesequences of these N-addition analogues may be composed of all D- or allL-amino acids and the antimicrobial activity of the resulting all D- orall L-oligopeptides will be the identical to each other.

It is contemplated that antimicrobial peptides attaining secondarystructure similar to that of the peptides of the instant invention maybe modified for the purpose of covalent attachment to a polymer.Accordingly the present invention provides a series of non-naturalpeptides useful as precursors for covalent incorporation into a polymer.These peptides can be incorporated into the polymer as outlined in FIG.3. Upon subsequent deprotection of the modified peptide a peptide-boundpolymer that is useful as an antimicrobial agent will result. Any of thepeptides described in this invention as well as other antimicrobialpeptides which share similar structural features (e.g., magainins,cecropins) may be modified at the N-terminus. The side chains of theamino acids in any of these antimicrobial sequences are appropriatelyprotected with labile protecting or blocking groups.

It is also contemplated that the introduction of one of a number ofactivated functionalities (e.g., vinyl groups, bis or monoalkylamines)via a spacer group of a suitable length would be possible withoutsignificantly altering the antimicrobial activity of the subsequentlyformed polymer after full removal of the peptide side-chain protectinggroups. Thus, the nature of the pendant modifying group is not limitedin this invention. For the purpose of the present invention, but not tobe limiting, suitable N-terminal modifying groups may be selected fromthe group comprising Lys, Lys(e-Boc), Lys(a-Boc), acyl halide, epoxy,carboxy, isocyanate, hydroxyl, alkenyl halide and acryloyl.

Peptide synthesis

The oligopeptides of the present invention may be prepared by any methodknown in the art, but generally will be prepared by manual or automatedsolid phase peptide synthesis (SPPS). An automated solid phase peptidesynthesis method is preferred because it is readily adaptable to largescale commercial production.

The concept of SPPS is known in the art and has been outlined in FIG. 1.Briefly, an N-α-derivatized amino acid (AA1) is attached to an insolublesolid (R) support via a linker. The amino acid may be attached directlyto the linker-support or first attached to the linker, with subsequentattachment of the amino acid-linker to the support. The N-a-blockinggroup (T) is then removed (deprotected) and the aminoacid-linker-support is thoroughly washed with solvent. The next aminoacid (AA2) (which is N-α-X-protected) is then coupled to the aminoacid-linker-support as either a preactivated species (i.e., symmetricalanhydride, active ester) or directly (in situ) in the presence ofactivator. Coupling may also be carried out using peptides instead ofsingle amino acids, where the peptide is N-α and side chain protected.After this reaction is complete, the N-α-dipeptide (oroligopeptide)-linker support is washed with solvent. Thedeprotection/coupling cycle is repeated until the desired sequence ofamino acids is generated. The peptide-linker-support is cleaved toobtain the peptide as a free acid or amide, depending on the chemicalnature of the linker. Ideally, the cleavage reagent also removes theamino acid side chain protecting groups, (◯) which are stable to thedeprotection reagent. These steps may be carried out as either a batchprocess, where the support is filtered between each step, or as acontinuous flow process, where the support is always solvated duringreagent exchange.

Currently there are a wide variety of resin supports commerciallyavailable for SPPS. A suitable support must be in particle or a physicalsize and shape that will permit ready manipulation and rapid filtrationfrom liquids, and yet be inert to all reagents and solvents used duringthe synthesis of the peptide. For the purposed of the present inventionsuitable resin supports include but are not limited to cross-linkedpolystyrene and polyamide resins where the polystyrene resin bearing thetrade mark SASRIN (BaChem Biosciences, Ltd.) is most preferred.

Although butyloxycarbonyl (Boc) is an effective blocking group itrequires repetitive acidolysis for deprotection which may disrupt acidsensitive peptide bonds and give rise to catalyzed side reactions. Amore acid labile N-α-amino blocking group is the base labile9-fluorenylmethoxycarbonyl (Fmoc) group. Fmoc may be bonded to theα-amino group of an amino acid. Where the first amino acid to beattached to the resin is Fmoc protected it may be attached via an acidsensitive linker employing a coupling agent diisopropyl carbodiimide(DIC) and non-nucleophilic bases. Fmoc protected amino acids maysubsequently be deprotected by base (usually piperidine). The secondFmoc amino acid is then coupled as either a preactivated species (i.e.,ester or symmetrical anhydride) or without preactivation (in situ),where several different activators may be utilized. After the desiredpeptide sequence has been obtained, the peptide-support and the basestable side chain blocking groups are acid (usually TFA) cleaved. Areview of typical solid phase peptide synthesis methods is given by J.M. Stewart, J. D. Young, in Solid Phase Peptide Synthesis, 2nd Edit.,Pierce Chemical Co., 1984).

In a preferred embodiment peptide syntheses employs a sequentialstrategy using Fmoc-blocked alpha amino acids, an acid sensitive resin,(SASRIN) Boc-blocked epsilon lysine amino acids and common deprotectionand coupling chemistry, known to one skilled in the art of peptidesynthesis. Initially the polystyrene resin is activated with theattachment of an acid linker. Coupling of the first amino acid to thesolid support is performed with diisopropylcarbodiimide,dimethylaminopyridine and N-methyl-morpholine. Attachment of the secondand remaining amino acids in the sequence is achieved through removal ofthe Fmoc blocking group with pipiridine followed by the coupling stepwith Castro's reagent (benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate), anddiisopropyl-ethylamine. Deprotection and coupling are repeated for eachsubsequent amino acid to be added until the peptide synthesis iscomplete. The final peptide product may be deprotected with and cleavedfrom the resin with 25% TFA and neutralized in pyridine forincorporation into polymer compositions. Alternatively, the peptide maybe cleaved from the resin in protected form with 1% TFA for use as asoluble antimicrobial.

For the purpose of permanent covalently bonding of the peptide to apolymer, an N-terminal modifying group comprising either a monoamine orbisamine derivatized blocked peptide may be constructed. Attaching theterminal modifying group to either the core or one of the N-additionanalogues is achieved either (a) by the employment of the solid phasemethod described for the previous amino acid residues or (b) by solutionreaction of the cleaved side-chain protected peptide with an appropriatecompound via either the free amino or carboxy terminus.

One possible scheme for the construction of monoamine and bisaminederivatized peptides is illustrated in FIG. 2. Preparation of anN-terminal Fmoc protected peptide may be prepared by solid phase peptidesynthesis as described above. Before cleavage from the resin the peptideis deprotected in standard fashion with piperidine and coupled to eitheran Fmoc-Lys-Boc or Fmoc-Lys-Fmoc activated amino acid with standardcoupling reagents used in SPPS. Following Fmoc deprotection and cleavagefrom the resin with TFA the Fmoc-Lys-Boc addition gives rise to themonoamine peptide derivative while the Fmoc-Lys-Fmoc addition give riseto the bisamine peptide derivative. Cleavage with 25% TFA will give thefully deprotected oligopeptide, whereas treatment with 1% TFA will givelysyl-side chain-blocked oligopeptides. It is further contemplated thata more exhaustive one-step solid phase synthetic scheme could beemployed to prepare oligopeptides that are of higher purity as taught byFunakoshi, S. et al., Proc. Natl. Acad. Sci. USA, 88, 6981-6985, 1991).It is also contemplated that peptide monomers containing a pendant vinylfunctional group for polymerizing into polymers can be prepared by SPPSemploying the method described by Matsuda et al (JP 3291298)

It is contemplated that a variety of other functional peptidederivatives may be constructed where specific modifications andvariations can be produced by methods well known to those skilled in theart of organic synthesis.

Although specific amino acid sequences have been defined, one ofordinary skill in the art will realize that this invention provides abasic amino acid sequence upon which many modifications and variationsare possible, provided that the juxtaposition and total number ofhydrophobic and basic hydrophilic amino acids falls within thelimitations disclosed herein. These limitations do not preclude thepossibility that the oligopeptides of the instant invention may also beproduced by recombinant organisms by methods well known to those skilledin the art of genetic engineering.

Peptide-Polymer Composition

The present invention also provides a product composition comprising aneffective antimicrobial peptide sequence incorporated into a polymer.The polymer may be natural or synthetic and may be selected from thegroup comprising polyesters, polyamides (e.g., Nylons 6, 66, 11, or610), polyurethanes, polyolefins, polyacrylates, polysaccharides (e.g.,chitin, cellulose, or algenate), polyamide coated diatomaceous earth,cellulosics, silks, nylons, biopolymers (e.g., elastin, collagen, orzein) or any polymer of synthetic or biological origin that can beformed into an object and that allows contact of the oligopeptide withthe microorganism within the matrix of the polymer. For the purposes ofthe present invention polymers containing either silk, polyamides,polystyrenes or polyurethanes are preferred. The antimicrobial peptidesequence usually constitutes the minor component of the composition andcan be present in amounts ranging from 1 to 15 percent based on weight.It is contemplated that any antimicrobial peptide sequence may beincorporated into the preferred polymers where peptides that formamphiphilic helices are preferred. Examples of such peptides may includebut are not limited to, the core oligopeptide, the N-addition analoguesof the core oligopeptide, as well as the naturally ocurring magainins,cecropins, protamines and their derivatives.

Preparation of Peptide-Polymer Composition

Incorporation of the bioactive peptide or oligopeptide may beaccomplished either by a process of coating or spinning effectiveamounts of the peptide onto the desired polymer or by a reactive processresulting in the covalent bonding of the peptide to the polymer.

Active preparations of the oligopeptide and polymer involving a coatingprocess can be prepared in a film or fiber form by one of severalmethods. In one embodiment, active film or fiber preparations can bemade by coating the shaped object with the oligopeptide from an aqueoussolution containing from about 1 to 15 weight percent of theoligopeptide. The coating solutions may also contain other smallwater-soluble molecules such as salts that will neither augment norinterfere with the antimicrobial action of the oligopeptide.

In another embodiment, active preparations of the antimicrobialoligopeptide and polymer can also be made by preparing a solution ormixture of the oligopeptide and the polymer, (hereinafter referred to asa "dope mixture"), and casting or forming the shaped article, fiber orfilm from the dope mixture. The shaped article, fiber or film containingthe oligopeptide may be quenched in a suitable non-solvent. For example,dope mixtures comprising polyurethane and the core oligopeptide may bequenched in water or methanol. The shaped article, fiber or filmcontaining the core oligopeptide may also be formed by allowing theshaped article to dry in air or under a suitable atmosphere to preventundesirable oxidative reactions.

In another preferred embodiment the present invention also provides amethod of preparing a drawn silk fiber with the oligopeptide by aprocess that involves: solubilizing the oligopeptide in a solvent;mixing the oligopeptide solution with a solution of natural or anunnatural silk in the same solvent; forming the fiber from a microscalespinning apparatus; and drawing the fiber to its maximum extension overa 24 hour period followed by drying. The solvent must be inert so as tocause minimal chemical changes to the composition. Solvents for thesolubilization of the oligopeptide may include any inert solvent orcombinations of inert solvents with power to mutually dissolve theoligopeptide and the polymer to high enough concentrations such that thefinal working dope solution is viscous enough to permit the drawing orfibers or making of films. For the purposes of the present inventionprefered solvents include hexafluoroisopropanol or 70 weight percentlithium thiocyanate in water.

Forming the fiber composition of the oligopeptide and silk may beaccomplished by any method known to one of ordinary skill in the art,however the process that is most preferred is a microscale spinningprocess described by Lock et al. in commonly owned U.S. Ser. No.07/827,141 herein incorporated by reference. In the preparation of theoligopeptide-silk fiber composition it is desirable to prepare a fiberwith a low denier. The lower denier fiber incorporates the advantage ofachieving a "tighter" weave and better barrier against the penetrationof infectious agents or fluids as well as a larger surface area forcoating of effective bactericidal or antiviral agents. This largersurface area increases the likelihood that the anti-microbial agent willeffectively present itself against the contaminating medium.

A silk fiber with lower denier or a smaller radial thickness is preparedwith increasing quantities of the oligopeptide in the dope. Levels ofoligopeptide present at 2.5 to 10 percent based on weight will effect alarger fiber draw and decrease the denier 20 to 30 percent.

In an alternative embodiment the peptides of the instant invention maybe covalently incorporated into polymer compositions. As is known tothose skilled in synthetic polymer chemistry, once a polymer or oligomersuch as a polypeptide that has one functional group or two functionalgroups at one end of the polypeptide, the polypeptide can beincorporated into a synthetic (nonpolypeptide) polymer as a pendantgroup, also called a side chain. The synthesis of such polypeptides isdescribed above. It is preferred that there not be any other functionalgroups on the polypeptide that can take part in the reaction which joinsthe polypeptide to the synthetic polymer, or a monomer which will beused to make a synthetic polymer. If such other functional groups arepresent, the polypeptide may serve to crosslink the synthetic polymer,thereby not being simply a side chain. Preferred functional groups to beplaced on the end of the polypeptide are amino and hydroxy.

If one functional group is present at the end of the polypeptide, thepolypeptide can be joined to the synthetic polymer in the two followingways. The polypeptide can be bonded to a molecule which can later act asa monomer which forms a synthetic polymer. For example, a polypeptidewith a hydroxyl or amino end can be reacted with an isocyanatecontaining monomer such as 2-isocyanatoethyl methacrylate, the hydroxylor amino groups reacting with the isocyanate group to form a urethane ora urea, respectively. The resulting methacrylate can then be freeradically polymerized by itself or copolymerized with other freeradically polymerizable monomers such as other (meth)acrylates orstyrene, to form a copolymer. The polypeptide will be a side chain onthis synthetic polymer. Alternately, the 2-isocyanatoethyl methacrylatecan be (co)polymerized to form a synthetic polymer, and then the amineor hydroxy terminated polypeptide can be reacted with the isocynategroups in the polymer, so that the polypeptide again becomes a sidechain on the synthetic polymer. Similar reactions on terminal mono-functional polypeptides can be carried out with other functional groups(for instance, epoxy) present in monomers for synthetic polymers, or inthe synthetic polymers themselves.

Terminally difunctional polypeptides can also become side chains insynthetic polymers. For instance, these difunctional polypeptides can beused as monomers in certain condensation polymerizations, particularlywhen one or more of the functional groups participating in the formationof the synthetic polymer are also the functional groups on the end ofthe polypeptide. For example, when the two functional groups on the endof the polypeptide are amino, reaction with diacyl halides (andoptionally and preferably other diamines) will yield a polyamide withpendant polypeptide groups. If the functional groups on the polypeptideare hydroxyl, reaction with diacyl halides (and preferably other diols)will lead to a polyester. Reaction of ends having 2 amine or 2 hydroxylgroups with a diisocyanate, or a so-called urethane prepolymer, willlead to a polyurea, or a poly(urethane-urea), or a polyurethane withpendant polypeptide groups. Given the relatively low thermal stabilityof polypeptides compared to many synthetic polymers, polymerizationsthat can be carried out a relatively low temperatures are preferred.

As those skilled in synthetic polymers realize, with the variety offunctional groups available on the ends of these polypeptides, the abovedescription merely gives some of the possibilities for making syntheticpolymers with pendant polypeptide groups, both, from the point of viewof the type of synthetic polymer made, and also the reactions used toattach the polypeptide to a synthetic polymer, or monomers used to makea synthetic polymer.

In another embodiment, bioactive peptides may be covalently attached topolymer compositions as side chain moieties by first modifying asuitable support with a non-cleavable linker followed by attachment ofthe carboxyl terminal amino acid to the linker, elongation of thepeptide chain by repeating the steps in solid phase peptide synthesis tosynthesize the full antimicrobial peptide sequence and finally removalof all side chain protecting groups. One specific instance of thisembodiment is outlined in FIG. 3. Here, a polyamide resin is modified bythe addition of ethylenediamine (EDA) to provide a resin with an aminofunctional group. Sequential additions of N-α-Fmoc and N-ε-Boc blockedamino acids may then be made using the coupling and deprotectionchemistry of SPPS as outlined above.

Alternatively, when a non-specific prederivatized support is employed,then several different peptides may be reacted simultaneously to createa support with broad spectrum biocidal activity. Conversely, when thepeptide-support is prepared by conventional strategy as used in solidphase peptide synthesis except that a non-hydrolyzable linkage iscreated between peptide and the support, only one type of antimicrobialpeptide may be attached to the support. The latter approach has theadvantage of providing a very specific and direct approach fordetermining whether the antimicrobial peptide is effective when attachedpendant to a polymer and restricted to surface penetration of themicro-organism.

Demonstration of Antimicrobial Activity

Determination of antimicrobial activity of the core oligopeptide, theN-addition analogues as well as the polymer compositions containingthese oligopeptides may be accomplished by standard techniques. For thepurposes of the present invention Eschericia coli or S. aureus arepreferred.

All manipulations with microorganisms involved the application ofstandard sterile techniques and materials commonly employed inmicrobiology. All cells were grown at 37° C. and cell concentrationswere measured by optical density measurements at 600 nm in a PerkinElmer Model 552 UV/VIS spectrophotometer. For determination ofantimicrobial activity of soluble oligopeptides, stationary cultures arepreferred whereas culture tubes or shake flasks are preferred when thecompositions are insoluble in aqueous media.

Typically, assays for antimicrobial activity of soluble oligopeptidecompositions were carried out in microtiter wells. Each well was chargedwith 150 μl of LB broth containing cells at a final concentration ofbetween 1×10⁶ to 1×10⁷ cells/ml and oligopeptide at final concentrationsof 1×10⁻³ to 1×10⁻⁶ M. It is preferred that the oligopeptides bedissolved in water prior to addition to the assay medium and that cellsbe inoculated from mid-log phase cultures. Cells were incubated at 37°C. until reaching a cell density corresponding to an absorbance readingof 0.1 A at 600 nm, at which point periodic absorbance readings weretaken to determine which cultures demonstrated cell growth inhibition.Cultures showing cell growth inhibition were incubated for an additional15 hr. at which time a final absorbance reading was taken. FIGS. 4a-dillustrate the cell growth inhibition of E. coli cultures incubated inthe presence of various oligopeptides.

In situations where the oligopeptide compositions were insoluble inaqueous media, a culture tube or shake flask culture method ispreferred. Oligopeptide compositions comprising a polymer or a fiber areexamples of such insoluble oligopeptide compositions.

Insoluble oligopeptide compositions were prepared as described above.Sterile culture tubes containing about 5 ml of LB broth were inoculatedwith cells and oligopeptide compositions. The preferred final celldensity for this assay is between 1×10⁶ to 1×10⁷ cells/ml where cellsare inoculated from mid-log phase cultures. Weight percent ofoligopeptide in the insoluble compositions were on the order of 0.018%to 15%. At periodic intervals aliquots were removed from the culturetube and transferred either to a disposable semi-microcuvette or to amicrotiter plate cell for absorbance readings on a uv/vis spectrometerat 600 nm or the microplate reader at 590 nm, respectively. FIG. 5illustrates cell growth inhibition, plotted as absorbance at 600 nm as afunction of time. Although any amount of oligopeptide may be present inthe insoluble oligopeptide compositions it is preferred that theoligopeptide be present at concentrations greater than or equal to 10%by weight of the total compositions.

Occasionally, a shake flask method was employed for insoluble articles.In the shake flask method, 75 mL of 0.6 mM phosphate buffer, pH 7.2 and0.100 mL of the stock cell culture and the oligopeptide sample wereadded to a 250 mL polystyrene tissue culture flask. The flasks wereincubated in a shaking water bath at 37° C. At specified times (1 hourand 24 hours), an aliquot (0.100 mL) was removed from the shake flaskand serial dilutions ranging from 10 to 1000-fold were made to allow theenumeration of cells on agar plates. Each agar plate was inoculated with0.200 mL of the diluted cell suspension, and the suspension wasuniformly spread on the plate. Colony-forming units CFU on the agarplates were counted after 24 hours in an incubator (37° C.). Cells wereidentified by visual observation and use of a colony counter. TotalCFU/mL was determined by correcting the cell count for the dilutionfactor.

The present invention provides a method of killing microorganisms withthe oligopeptides and water-insoluble films, fibers or fabricscontaining the antimicrobial oligopeptides. The oligopeptide or theshaped articles containing the oligopeptide is placed in contact withsolutions containing microorganisms at concentrations up to 10⁸ /mL withall essential growth nutrients for the microorganism. Contact times of 1to 24 hours in media at temperatures in the range of ambient and 37° C.are effective in inhibiting growth of all gram negative bacteria, grampositive bacteria and yeasts present at concentrations equal to and lessthan 10⁸ /mL.

As has been mentioned the peptides of the instant invention rely ontheir unique amphiphilic secondary structure for their antimicrobialactivity. Although the entire mechanism of action for antimicrobialpeptides forming amphiphilic helices is unknown, several partialexplanations have been put forth in the art. Juretic et al. (FEBS, 249,219-223, (1989)) and Westerhoff et al. (Proc. Natl. Acad. Sci, USA, 86,6597-6601, (1989)) teach that the mechanism of action of magainin andits analogues is membrane depolarization to prevent respiration oroxidative metabolism. The site of oxidative metabolism is the innermembrane of the microorganism. Zasloff (WO 9004408) teaches that thepeptides must be of sufficient length (>20 peptide residues) to span thelipid bilayer membrane to cause membrane perturbation. Zasloff alsoteaches that the activity of these peptides is potentiated in thepresence of toxic anions, which supports the suggestion that ion channelformation is the mode of microbial killing. Cruciani et al., (Biophys.J. 53, 9a, (1988)) demonstrated that these peptides form anion-selectivechannels with synthetic lipid bilayers and Urritia et al., (FEBS 247,17-21, (1989)) suggests that ion channel formation by antimicrobialpeptides occurs by aggregation of the peptides. One would expect that anantimicrobial peptide which relies on its amphiphilic secondarystructure for bioactivity such as magainin or the peptides of theinstant invention would be precluded from forming such self-associated,multimeric channels when the peptide is covalently bound to a rigidsupport. Thus it is highly unexpected that such a peptide could becovalently bonded to a polymeric support and still retain its biologicalactivity.

The following non-limiting examples are meant to illustrate andexemplify the invention but are not meant to limit it in any way.

EXAMPLES General Methods

The growth medium may be common commercial preparations such as LB brothor containing yeast extract, Tryptone or hydrolyzed protein, sodiumchloride in water. Other growth media may be used and the appropriatemedium for growth of the particular microorganism will be known bysomeone skilled in the art of microbiology. Solutions containing none orincomplete nutrients for growth may be sterile saline, distilled wateror solutions containing other salts or ingredients dissolved in salineor water and this final solution is not sufficient to support growth ofmicro-organisms but is also non-lethal to the microorganisms.

All peptides were synthesised using the technique of solid phase peptidesynthesis (SPPS) and employing methods and materials well known to thoseof skill in the art. All amino acids used were purchased in protectedform from either BaChem Biosciences, Ltd. (Philidelphia, Pa.) orMillipore Corp. (Boston, Mass.) Protocols for SPPS are well known in theart and descriptions may be found in Stewart et al Solid Phase PeptideSynthesis, 2nd Edit, J. M. Stewart, J. D. Young, 1984, Pierce Chem. Co.pp 18-27 or G. A. Grant, Synthetic Peptides: A Users Guide, Freeman, NY,pp 77-260 herein incorporated by reference.

Example 1 Preparation of Oligopeptides

Peptide synthesis of Formulae I-IV (Table I) was accomplished usingmanual solid phase peptide synthesis (SPPS). The strategy employed theuse of Fmoc-amino acids and a super acid-sensitive resin to prepare thepeptides I-IV and their fully protected analogues. All reagents andsolvents were of analytical purity or HPLC grade, respectively, and wereused without further purification. Alpha-amino-Fmoc-protected aminoacids (Bachem Biosciences, Philadelphia, Pa.) were used. The epsilonamino side chain of Lysine was protected with the grouptert-butyloxycarbonyl (Boc). The solid phase support was SASRIN resin(2-methyoxy-4-alkoxy-benzyl alcohol derivative of a crosslinkedpolystyrene; Bachem Biosciences, substitution level=0.9 mequiv/gm). Thefirst amino acid was anchored to the resin using diisopropylcarbodiimide (DIC) in the presence of dimethylaminopyridine (DMAP) andN-methyl-morpholine (NMM). Subsequent amino acids were added to theresin with the reagent that forms in situ active esters.

In a 50-mL round-bottomed flask, DIC (1.56 mL, 10 mmol) was added to acooled solution of Fmoc-Leu-OH (3.53 g, 10 mmol) dissolved in 30 mLdichloromethane (DCM). The solution was stirred for 10' and theice/water bath was removed and the solution was allowed to stir foranother 10' before the addition of DMAP (61 mg, 0.5 mmol) and NMM (0.293mL, 2.67 mmol). The reaction mixture was transferred to a 500-mL SPPSvessel that contained 2.3 g of SASRIN resin (2.0 mequiv). The flask wasrinsed with an additional 20 mL DCM and the mixture was allowed to stirovernight. The soluble components were flushed and the resin was washed:DCM (4X); methanol, MeOH (3X); dimethylformamide (DMF, 3x); DCM (3X) andMeOH (3X). The resin clump was transferred to a preweighed coarsefritted funnel, washed with MeOH and allowed to dry to constant weightto determine yield. Yield=82%. The unreacted sites on the resin arecapped by reaction with acetic anhydride. The resin is transferred backinto the SPPS vessel and suspended in 20-30 mL DCM. In a separate flask,the following are mixed: acetic anhydride (0.47 mL, 5 mmol);N,N'-diisopropylethylamine (DIEA; 4.0 mL of 0.5M solution in DCM; 2.0mmol); DCM (15 mL). This acetylating mixture is added to the resinsuspension and allowed to react for 30' before washing (DCM, 3X; MeOH,3X; DMF, 3X). Sequential coupling of the amino acids in the sequence asperformed by a repeat of the following procedure. The terminal aminoacid was deprotected by suspending the resin in 20-30 mL of 20 or 50%(v/v) piperidine in DMF. Deprotection reactions were run for 20' beforedraining the piperidine and washing the resin. Coupling was initiated bythe addition of a solution containing: the next Fmoc-amino acid (at a2.5 to 6-fold excess over the mequiv on the resin); BOP (5 to 12-foldexcess over the mequiv on the resin) in sufficient solvent so that themolar concentration of Fmoc amino acid is about 0.25M. Following theaddition of this solution is the addition of DIEA (5 to 12 fold excessover the mequive on the resin) and a rinse with the coupling solvent(DMAc) such that the final concentration of the activated amino acid isaround 0.1M. The reaction is allowed to proceed for 2 to 2.5 hours atambient temperature. The reaction mixture is washed (DMAc, 3X; MeOH, 3X;DMF, 3X) and deprotection and coupling are repeated for each subsequentamino acid to be added until the peptide synthesis is complete.

If the desired final peptide product is a deprotected peptide,deprotection and cleavage of the peptide from the resin may beaccomplished with 25% TFA and the peptide neutralized in pyridine forincorporation into polymer compositions. Thus, after synthesis and Fmocdeprotection of the N-terminal amino acid, the completely deprotectedoligopeptide was cleaved from the resin by treating the resin threetimes with 25% trifluoroacetic acid in methylene chloride.

The effluent was collected into a receiving flask sitting in anice/water bath at 4° C. containing a stoichiometric quantity of pyridinefor neutralization.

Solvent was removed by rotary evaporation without using heat. The fullydeprotected oligopeptides were precipitated by addition of cold,anhydrous diethyl ether to the oil. The oligopeptide was collected as acrude white solid by filtration, vacuum dried, then redissolved in 10%acetic acid. This aqueous solution was stripped of solvent on a rotaryevacuator.

Alternatively, if the desired final peptide product is a fully protectedpeptide, the peptide may be cleaved from the resin in protected formwith 1% TFA for use as a soluble antimicrobial. Thus, the fullyprotected oligopeptides were cleaved from the resin by treating theresin three times with 1% trifluoroacetic acid in methylene chloride.The effluent was collected in the same manner as for the deprotectedoligopeptides. After the solvent was removed, the protectedoligopeptides were precipitated by addition of cold, distilled water tothe oil. The flask containing the suspension sat on ice/water bath forseveral hours before collection of the solid by filtration.

For analysis, the oligopeptides were solubilized with 0.1%trifluoroacetic acid and analyzed by HPLC on an analytic reversed phasecolumn (BioRad HiPore 318, 4.6 mm×25 cm), using a Waters Millennium 2010HPLC system composed of two Model 510 pumps, Model 490E multiwavelengthdetector, Model 717 autosampler, column heater and a Millennium 2010chromatography manager with a pump control module. The peptides wereeluted by a linear gradient composed of buffer A, 0.1% trifluoroaceticacid in water, and buffer B, 0.1% trifluoracetic acid in CH3CN/H20,90/10. The gradient was run from 0 to 100% buffer B in 100 min with aflow rate of 1.0 mL/min at ambient temperature. The peptides weredetected at 220 nm.

Preparative purification of the peptides was accomplished on a WatersDelta Prep 3000 System using a Vydac C4 reversed phase column (22 mm×25cm) (The Nest Group; Southboro, Mass.). Buffer and gradient conditionswere the same as described for the analytical separation with theexception of the flow rate which was at 10 ml/min. Amino acidcompositions were determined on a Beckman 6300 amino acid analyzerfollowing 6N acid hydrolysis of the peptides by standard methods.

Example 2 Tests of Antimicrobial Activity of Soluble Oligopeptides

All manipulations with microorganisms involved the application ofstandard sterile techniques employed in microbiology. All cells weregrown at 37° C. Cell concentration was measured by optical densitymeasurements at 600 nm in a Perkin Elmer Model 552 UV/VISspectrophotometer. The microorganisms used were strains of E. coli. andS. aureus. On the day prior to the assay, an overnight culture wasprepared. On the following morning, the stationary phase cells weresubcultured and allowed to grow to mid-log phase.

Each well was charged with LB Broth (150 mcL). The oligopeptide sampleswere dissolved in water and were added to achieve the appropriate finaloligopeptide concentrations (ranging from 1×10⁻³ to 1×10⁻⁶ M) Finally,cells suspended in LB medium were added to a final concentration ofbetween 1×10⁶ to 1×10⁷ cells/mL. The microtiter plate was covered andallowed to gently rotate in an orbital shaker. When the cell growth wassuch that the optical density at 600 nm was in the range of 0.1 A,microtiter plate readings were taken at twenty to thirty minuteintervals until the optical density exceeded 1.2 A. Cultures thatdemonstrated cell growth inhibition by the oligopeptides were allowed togrow overnight for final readings approximately fifteen hours later.Control samples used for all experiments were: (a) LB brothonly=uninoculated control; (b) LB broth only+cells=inoculated control.

Data demonstrating cell growth inhibition of E. coli cultures isillustrated in FIG. 4a-d and in Table II. In FIGS. 4a-d cell growthinhibition is plotted as molar concentration of antimicrobial peptide asa function of the number of cells killed/ml. FIG. 4a-d illustrates theactivity of the core oligopeptide, the N-addition analogue of formulaII, the N-addition analogue of formula III, and the N-addition analogueof formula IV, respectively. All cultures were actively growing inlog-phase and at a cell concentration of 2.0×10⁷ cells/ml. As can beseen by the data the N-addition analogues have between 10 and 100 foldgreater activity against E. coli than does the core oligopeptide.

                  TABLE II                                                        ______________________________________                                                    Minimal Inhibitory Concentration μg/ml                         Peptide     E. coli      S. aureus                                            ______________________________________                                        Formula I   15.6         31.2                                                 Formula II  7.8          15.6                                                 Formula III 7.8          62.5                                                 Formula IV  7.8          15.6                                                 ______________________________________                                    

The data in Table II shows the bactericidal activity of the solublepeptides definded by the formula illustrated in Table I. Formula Icorresponds to the core oligopeptide, whereas formulae II-IV correspondto the N-addition analogues. It is evident from the data in Table IIthat the soluble oligopeptides exhibited antimicrobial activity againstboth gram positive and gram negative bacteria and that there is asignificant and unexpected increase in activity of the N-additionanalogues over the core-oligopeptide.

Example 3 Preparation of Silk Fibroin/Oligopeptide Films

Solutions of 20% w/v silk fibroin in hexafluoroisopropanol (HFIP)(Aldrich Chemical Co., Milwaukee, Wis.) were prepared. These 20%solutions were diluted to a final concentration of 10% silk by addingaliquots of a core oligopeptide stock solution in HFIP. Four coreoligopeptide/silk solutions were made corresponding to 0%, 0.018%, 0.18%and 15% oligopeptide in the composition.

Films were cast by dropping the solutions onto glass coverslips, thenquenching in a large methanol bath. The films were washed extensively inwater to remove traces of the organic solvents. The weights of the filmsamples were typically 3 to 5 mg, thus the amount of oligopeptide ineach sample ranged from 3 mcg to 300 mcg. The films were used as is inthe tests of antimicrobial activity.

Example 4 Preparation of Fibers from Oligopeptide and Silk Blends

Three different dope solutions were prepared containing 500 mg silkfibroin prepared as described by Lock in U.S. Ser. No. 07/827,141 hereinincorporated by reference, 3 mL HFIP and 0, 13, or 56 mg of the coreoligopeptide. The final percentage of core oligopeptide of the totalsolids in the dope solution was 0, 2.5 and 10 percent, respectively. Thedope solutions were placed in small polyethylene bags, sealed thenkneaded to remove any undissolved polymer or polypeptide particles. Thebag containing the dope was centrifuged to remove any dissolved gases.The dope was then placed into a microscale spinning apparatus and fiberwas formed by the process described by Lock in U.S. Ser. No. 07/827,141herein incorporated by reference.

After fiber preparation, the fibers were drawn by hand while still wetwith methanol (or the coagulation solvent) to their maximum extension.The drawing was performed during the 24 hour period immediatelyfollowing the preparation and drying of the fiber. As illustrated by thedata shown in Table III, the oligopeptide acts as a processing aid inthe drawing of the silk fibers by permitting drawing of the fibers to alonger, total elongation. Table III shows the physical properties ofdenier, tensile, elongation and modulus of the fibers drawn containing0, 2.5 and 10 percent of the core oligopeptide.

As can be seen by the data in Table III the denier for the silk/2.5% LKPand the silk/10% LKP is 24% and 41% smaller respectively than that ofsilk alone. Table III further shows that the other desirable parametersof silk fiber such as tensile, and modulus are not affected by thepresence of the core oligopeptide.

                  TABLE III                                                       ______________________________________                                        Physical Properties of the                                                    LKP/Silk Fibroin Co-Spun Fibers                                               Sample   Denier   Y.O.F.   Tensil Elong. Mod.                                 ______________________________________                                        silk fibroin                                                                           905      3.5 yds. 2.04   47.8   41.8                                 silk/2.5% LKP                                                                          728      4.5 yds. 2.22   27.8   47                                   silk/10% LKP                                                                           638      5.0 yds. 2.13   19.7   46.2                                 ______________________________________                                    

Example 5 Tests of Antimicrobial Activity of Silk/Core OligopeptideFilms

In situations where the peptide or test system were insoluble, a culturetube or shake flask method was employed to test for antimicrobialactivity. Antimicrobial activity of silk/core oligopeptide compositionwas tested in this manner.

Compositions of silk/core oligopeptide were prepared at varying percentcore oligopeptide to silk as described in Example 3. Seven 10 ml sterileculture tubes were filled with 5.5 mL LB media each. Experimental tubeswere inoculated with silk/core oligopeptide at varying percentage ofoligopeptide to silk along with E. coli cells. Of the seven tubes, onecontained LB medium only and one contained cells only. The silk/coreoligopeptide compositions tested had either 0%, 0.018%, 0.18% or 15%core oligopeptide by weight. E coli cells inoculated into the tubes weretaken from cultures actively growing at log-phase and were inoculated toa final concentration of between 1×10⁰⁶ to 1×10⁷ cells/mL. At periodicintervals aliquots were removed from the culture tube and transferredeither to a disposable semi-microcuvette or to a microtiter plate cellfor absorbance readings on a uv/vis spectrometer at 600 nm. FIG. 5illustrates cell growth inhibition, plotted as absorbance at 600 nm as afunction of time.

As can be seen by the data illustrated in FIG. 5, cultures containingthe silk/core oligopeptide at 15% oligopeptide demonstrated completecell growth inhibition, whereas silk/core oligopeptide compositionscontaining less than 10% oligopeptide were ineffective in inhibiting thegrowth of E. coli cells.

Example 6 Preparation of Bisamine Derivatized Blocked AntimicrobialPeptides

A peptide of Formula VI having a bisamine funtionl lysine at theN-terminus was prepared using chemistry familiar to one skilled in theart of SPPS.

Formula VI:

    Lys-Gly- Leu-LyS(Boc)Lys(Boc)-Leu-Leu-Lys(Boc)-Leu!.sub.2  (SEQ ID NO: 10)

Synthesis was accomplished up to the final N-terminal amino acid usingthe pattern of coupling and deprotection described in Example 1. Afteraddition of the N-terminal amino acid in any of the sequences informulaes I-IV, the terminal amino acid was coupled in standard fashionwith BOP-activated Fmoc-Lys(Fmoc). Following addition of thisbis-Fmoc-amine-protected lysine, the SASRIN resin was treated withpiperidine and washed. The Boc-protected oligopeptide was cleaved bytreating the resin three times with 1% trifluoroacetic acid in methylenechloride, for 10-15 minutes. Work-up and isolation of the purifiedblocked oligopeptide with a terminal lysine commenses as described forthe parent peptides in Example 1.

Example 7 Preparation of a Monoamine Derivatized Blocked AntimicrobialPeptide

Peptides of Formula VII or VIII having a monoamine funtional lysine atthe N-terminus were prepared using chemistry familiar to one skilled inthe art of SPPS.

Formula VII:

    Lys-NE(Boc)-Gly- Leu-Lys(Boc)Lys(Boc)-Leu-Leu-Lys(Boc)-Leu!2 (SEQ ID NO:11)

Formula VIII:

    Na(Boc)Lys-Gly- Leu-Lys(Boc)Lys(Boc)-Leu-Leu-Lys(Boc)-Leu!.sub.2(SEQ ID NO: 12)

The preparation of the peptide of Formula VII or VIII proceeds asdescribed for the parent formulae through the sequence of addition ofthe N-terminal amino acid. The terminal amino acid was coupled instandard fashion with Fmoc-Lys(Boc) or Boc(Lys)Fmoc. Treatment withpiperidine, washing and resin cleavage with 1% trifluoracetic acid inmethylene chloride, work-up and purification as described in Example 1yielded a blocked oligopeptide with a terminal lysine with either a freealpha amine or a free epsilon amine.

Example 8 Preparation of Peptides Covalently Bound to Polyamide-ModifiedKieselguhr Resins

The methylester-activated polymer support PEPSYN K (Millipore Co.,Boston Mass.) was first modified by reaction with neat ethylene diamine24-48 hours at ambient temperature. The resin was washed in DMF (10X)and CH2C12 (10X), then vacuum dried. The resin was washed with 10%diethylamine in DMF, then reacted with BOP-activated Fmoc-Leucine (orthe carboxy-terminal amino acid of the sequence) to link the first aminoacid by the typical procedure outlined for solid phase peptidesynthesis. The coupling of subsequent amino acid residues proceeded asdescribed for the solid phase peptide synthesis (Example 1). Aftersynthesis and Fmoc deprotection of the N-terminal amino acid, the resinwas treated with 25% trifluoroacetic acid in methylene chloride toremove the Boc-protective group from all lysine residues in thesequence. The resin was washed with dilute (5%) pyridine in methylenechloride (3X), methylene chloride (10X), methanol (5X) and methylenechloride (10X), then vacuum dried. All control resins (i.e., resins withother chemical groups besides the antimicrobial peptide) were similarlytreated with the sequence of trifluoroacetic acid deprotection andwashing steps. The peptide bound resins were used without furthertreatment in the tests of antimicrobial activity. Composition and aminoacid sequence of the covalently bound peptides could be confirmed byperforming the entire solid phase peptide synthesis with a mixture ofthe non-acid sensitive ethylene-diamine modified resin and a typicalcleavable resin linker.

Example 9 Antimicrobial Activity of Peptides Covalently Bound toPolyamide Resin

Samples of peptides corresponding to the amino acid sequences SEQ IDNO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:1, werecovalently linked to the polyamide resin PepSyn K as described inExample 10 and were tested for antimicrobial activity. Five compositions(PEPSYNK-LKP, PEPSYNK-LKPGK, PEPSYNK-LKPG₂ K, PEPSYNK-LKPG₃ K, andPEPSYNK-KGLKP) were tested over a range of 0 to 5 mg/ml of oligopeptide.Since the covalently linked oligopeptide/polymer compositions wereinsoluble, the protocol for testing antimicrobial activity was modifiedfrom that for the soluble peptide compositions of Example 2. Here, ashake flask method was employed.

In the shake flask method, 75 mL of 0.6 mM phosphate buffer, pH 7.2 and0.100 mL of the stock cell culture and the oligopeptide samples, rangingfrom 0 to 5 mg/ml of oligopeptide were added to a 250 mL polystyrenetissue culture flask. The flasks were incubated in a shaking water bathat 37° C. At specified times (1 hour and 24 hours), an aliquot (0.100mL) was removed from the shake flask and serial dilutions were maderanging from 10 to 1000-fold to allow the enumeration or cells on agarplates. Each agar plate was inoculated with 0.200 mL of the diluted cellsuspension, and the suspension was uniformly spread on the plate.Colony-forming units CFU on the agar plates were counted after 24 hoursin an incubator (37° C.). Cells were identified by visual observationand use of a colony counter. Total CFU/mL was determined by correctingthe cell count for the dilution factor. In this manner the minimalinhibitory concentration (MIC) of oligopeptide for covalently linkedcompositions corresponding to PEPSYNK-LKP, PEPSYNK-LKPGK, PEPSYNK-LKPG₂K, PEPSYNK-LKPG₃ K, and PEPSYNK-KGLKP were determined.

In order to determine that the compositions were not cytotoxic ahemolysis assay was preformed. A 5.0% stock cell solution of humanerythrocytes in phosphate buffered saline (PBS) was prepared bysuspending 2.0 mL packed human erythrocytes in 38 mL of phosphatebuffered saline (10 mM sodium phosphate, 120 mM sodium chloride, 2.7 mMpotassium chloride, pH 7.5). Samples of the four compositions recitedabove were added to microfuge tubes containing 0.5 mL of cell solutionand 0.5 mL of PBS. Upon addition of sample, the vials were capped,gently homogenized, then allowed to gently shake in an incubator at 37°C. for 30 minutes. The samples were centrifuged to sediment the cells.The supernatants were transferred to cuvettes and the absorbances at 414nm were read in a Perkin Elmer 552 spectrophotometer. The 0 and 100%hemolysis controls were PBS and PBS +1.0% Triton X-100, respectively.

Table IV presents data depicting the minimal inhibitory concentration(MIC) of oligopeptide within each of the four covalently linkedoligopeptide/polymer compositions. As described in Example 8 allpeptides were covalently attached to the resin via a non-cleavablelinker, EDA. As can be seen by the data, covalent bonding of theantimicrobial peptides of Formula I-IV can be accomplished whilemaintaining the antimicrobial and non-cytotoxic properties of thepeptides.

                  TABLE IV                                                        ______________________________________                                        Covalently Linked                                                                            MIC (μg/mL)*                                                                           % Hemolysis at                                     Peptide/Polymer                                                                              E. coli     >6 mg/mL)*                                         ______________________________________                                        Pepsynk-LKP    807         0.9                                                PepsynK-LKPGK  2213        0.7                                                PepsynK-LKPG2K 1208        1.2                                                PepsynK-LKPG3K 938         12.7                                               PepsynK-KGLKP  888         >0.7                                               ______________________________________                                         *resin alone and resin with EDA linker without attached peptides gave no      antimicrobial or hemolytic activity                                      

Example 10 Regeneration of the Covalently Bound AntimicrobialPeptide-Polymers

Following use of the polyamide resin in an antimicrobial experiment, thesupernatant was carefully decanted from the solid material. The polymerwas separated from the original cell-containing media by suspension inautoclaved water (5X) and removal of the supernatant. The resin was thenwashed on a glass- fritted filter by the following: 0.02% aqueous sodiumazide (1X); sterile, distilled water (5X); methanol (5X) and methylenechloride (5X). The resin was vacuum dried overnight before reuse in anantimicrobial experiment.

Example 11 Preparation of Immobilized Magainins and their BactericidalActivities Against E. coli

Example 11 demonstrates that known bactericidal amphiphelic peptidessuch as magainin may be immobilized on a polymer and retain theiranti-microbial activity. The magainin 2-EDA-PepsynK resins were preparedusing a Milligen EXCELL® Automated Peptide Synthesizer employingFmoc-protected amino acids and BOP/HOBt with NMM to produce activeesters in DMF solvent. Side-chain protecting groups for specific aminoacids were: Ser(tBu); Lys(Boc); His(Trt); Glu(OtBu); and Asn(OPfp). Thesynthesis was started from Fmoc-Ser(tBu)-EDA-PepsynK resin (0.075 mmole;0.3 g, 0.25 mmol/g=substitution level). This starting resin was preparedby coupling the carboxy-terminal amino acid to EDA-modified PepsynK asdescribed in Example 8. The amino acid-EDA-PepsynK derivative (300 mg)was transferred to an empty EXCELL® reaction column and placed on theautomated synthesizer. The remaining amino acids of the desired peptidewere attached to the resin by repetitive deprotection, washing andcoupling steps and these steps follow the standard cycles used by theEXCELL® peptide synthesizer. At the end of the synthesis, the finalamino-terminal Fmoc group was removed prior to full deprotection. All ofthe protective groups were simultaneously cleaved by the washing of thefinal product in this cocktail: 95% trifluoroacetic acid; 5%thioanisole; 3% ethanedithiol and 2% anisole for 2 hr. Followingdeprotection, the peptide-resin was then washed extensively with thefollowing sequence of washes: 5X DMF; 5X DCM; 5X MeOH; 5X DMF; 5X DCM;5X MeOH and 5X diethyl ether. The resin was vacuum dried to remove finaltraces of cleavage cocktail or organic solvents to yield 490 mg ofmagainin 2-EDA-PepsynK. The amino acid composition was confirmed byperforming a control synthesis using as starting material a mixture of25% cleavable (Fmoc-Ser(tBu)--C(O)--O--Pepsyn KA) and 75% of theabove-described non-cleavable resin. At the end of synthesis and fulldeprotection, the cleaved peptide product was isolated, purified andcharacterized by amino acid composition and sequence analyses. Thesequence and compositions matched those of authentic magainin 2.

The derivatives of the 17-mer deletion peptide of magainin 2 and reversemagainin 2 peptide were prepared in the same manner as described abovefor magainin 2-EDA-PepsynK. The following amino acid derivatives wereprepared and used as starting materials for the indicatedpolymer-peptide product: Fmoc-valine, magainin2 17-mer deletion peptide;Fmoc-glycine, reverse magainin 2.

The bactericidal activity for each immobilized peptide was determined asdescribed in Example 9. The structures and the bactericidal activitiesof the immobilized peptides are shown in Table V. The relativeactivities against E. coli are consistent with what is known about thesoluble peptides. The 17-mer deletion peptide has activity comparable tothe full-length magainin. The reverse magainin is a much poorerbactericide than its normal sequence.

                                      TABLE V                                     __________________________________________________________________________    Name       Peptide Structure        % Reduction E. coli                       __________________________________________________________________________    EDA-PepsynK                                                                              S-N-M-I-E-G-V-F-A-K-G-F-K-K-A-S-H-L-F-K-G-I-G                                                            100%                                    magainin2  (SEQ ID NO:16)                                                     EDA-PepsynK magainin                                                                     V-F-A-K-G-F-K-K-A-S-H-L-F-K-G-I-G                                                                       99.3%                                    17-mer deletion                                                                          (SEQ ID NO:17)                                                     EDA-PepsynK reverse                                                                      G-I-G-K-F-L-H-S-A-K-K-F-G-K-A-F-V-G-E-I-M-N-S                                                          95.14%                                    magainin2  (SEQ ID NO:18)                                                     __________________________________________________________________________

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 18                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       LysGlyLeuLysLysLeuLeuLysLeuLeuLysLysLeuLeuLysLeu                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuLysLeuLeuLysLysLeuLeuLysLeuLeuLysLysLeuGlyLys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LysGlyGlyLeuLysLysLeuLeuLysLeuLeuLysLysLeuLeuLys                              151015                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuLysLeuLeuLysLysLeuLeuLysLeuLeuLysLysLeuGlyGly                              151015                                                                        Lys                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       LysGlyGlyGlyLeuLysLysLeuLeuLysLeuLeuLysLysLeuLeu                              151015                                                                        LysLeu                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuLysLeuLeuLysLysLeuLeuLysLeuLeuLysLysLeuGlyGly                              151015                                                                        GlyLys                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuLysLysLeuLeuLysLysLeuLysLysLeuLysLysLeuLeuLys                              151015                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuLysLysLeuLeuLysLeuLeuLysLysLeuLeuLysLeu                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       LeuLysLysLeuLeuLysLeu                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LysGlyLeuLysLysLeuLeuLysLeuLeuLysLysLeuLeuLysLeu                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      LysGlyLeuLysLysLeuLeuLysLeuLeuLysLysLeuLeuLysLeu                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      LysGlyLeuLysLysLeuLeuLysLeuLeuLysLysLeuLeuLysLeu                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      LeuLysLeuLeuLysLysLeuLeuLysLeuLeuLysLysLeuGlyLys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      LeuLysLeuLeuLysLysLeuLeuLysLeuLeuLysLysLeuGlyGly                              151015                                                                        Lys                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      LeuLysLeuLeuLysLysLeuLeuLysLeuLeuLysLysLeuGlyGly                              151015                                                                        GlyLys                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      SerAsnMetIleGluGlyValPheAlaLysGlyPheLysLysAlaSer                              151015                                                                        HisLeuPheLysGlyIleGly                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ValPheAlaLysGlyPheLysLysAlaSerHisLeuPheLysGlyIle                              151015                                                                        Gly                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GlyIleGlyLysPheLeuHisSerAlaLysLysPheGlyLysAlaPhe                              151015                                                                        ValGlyGluIleMetAsnSer                                                         20                                                                            __________________________________________________________________________

What is claimed is:
 1. A polymer-oligopeptide consisting of:(i) apolymer covalently linked to (ii) an antimicrobial oligopeptide capableof attaining an amphiphilic helical secondary structure and selectedfrom the group consisting of Lys Gly Leu Lys Lys Leu Leu Lys Leu Leu LysLys Leu Leu Lys Leu; (SEQ ID NO: 1), Leu Lys Leu Leu Lys Lys Leu Leu LysLeu Leu Lys Lys Leu Gly Lys: (SEQ ID NO:2), Lys Gly Gly Leu Lys Lys LeuLeu Lys Leu Leu Lys Lys Leu Leu Lys Leu; (SEQ ID NO:3), Leu Lys Leu LeuLys Lys Leu Leu Lys Leu Leu Lys Lys Leu Gly Gly Lys; (SEQ ID NO:4), LysGly Gly Gly Leu Lys Lys Leu Leu Lys Leu Leu Lys Lys Leu Leu Lys Leu;(SEQ ID NO:5), and Leu Lys Leu Leu Lys Lys Leu Leu Lys Leu Leu Lys LysLeu Gly Gly Gly Lys; (SEQ ID NO:6).
 2. The polymer-oligopeptide of claim1 wherein the polymer is selected from the group consisting ofpolyurethane, polyetherurethane, polyester, silicone, polyamide,polyolefin, polypeptide, polysaccharide, cellulosic, and silk.
 3. Thepolymer-oligopeptide of claim 2 wherein the polymer is a polyamide, apolystrene or a polyurethane.
 4. The polymer-oligopeptide of claim 2wherein the polymer is silk in fiber form.
 5. The polymer-oligopeptideof claim 3 wherein the oligopeptide is linked to the polymer by anon-cleavable linker.
 6. A process for inhibiting the growth ofmicroorganisms comprising the steps of:(i) contacting an effectiveamount of the polymer-oligopeptide of claim 1 with a population ofmicroorganisms; and (ii) leaving the polymer-oligopeptide in contactwith the population of microorganisms for a time sufficient to inhibitmicroorganism growth.